Essential oil isolation and characterisation

4.4.1.1. Plant material:

Lavandula angustifolia Mil. is not naturally occurring plant in Portugal, so it has to be artificially cultivated in fields. Flowers were provided by Ervital, a Portuguese plant producer. These plants were cultivated in Castro de Aire region. Fresh plants were harvested and then dried in the laboratory for two days, in an open space in the shadow.

4.4.1.2. Isolation:

Essential oil was obtained from flowering aerial parts of the plant by hydrodistillation for three hours. The distillation was performed according to European Pharmacopoeia [74] using Clevenger-type apparatus. The oil was obtained in yields of 2 % (v/w) and was stored in refrigerator to conserve it.

4.4.1.3. Analysis:

Analytical gas chromatography was carried out in Hewlett-Packard 6890 (Agilent Technologies, Palo Alto, CA, USA) gas chromatograph and HP GC ChemStation Rev. A.05.04 data handling system, equipped with a single injector and two flame ionization detection (FID) systems. A graphpack divider (Agilent Technologies part no. 5021-7148) was used for simultaneous sampling to two Supelco (Supelco, Bellefonte, PA, USA) fused silica capillary columns. Two different stationary phases were used: SPB-1 (polydimethylsiloxane 30 m x 0,20 mm i.d. with film thickness 0,20 µm) and SulpecoWax-10 (polyethyleneglycol 30 m x 0,20 mm i.d. with film thickness 0,20 µm). The oven program temperature was programmed to increase from 70 ˚C to 220 ˚C at 3 ˚C/min increments, 220 ˚C – 15 min) with injection temperature 250 ˚C. The carrier gas was helium adjusted to linear velocity of 30 cm/s and splitting ratio 1:40. The temperature of detectors was set for 250 ˚C.

Mass spectrometry analyses were carried out in a Hewlett-Packard 6890 gas chromatograph fitted with a HP-1 fused silica column (polydimethylsiloxane 30 m x

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0,25 mm i.d. film thickness 0,25 µm), interfaced with Hewlett-Packard mass-selective detector 5973 (Agilent technologies) operated by HP enhanced ChemStation software, version A.03.00. The same parameters as described above for gas chromatography were used. Interface temperature was set on 250 ˚C, MS source temperature was 230 ˚C, MS quadrupole temperature was 150 ˚C and ionization energy 70 eV, ionization current 60 µA and scan range 35 - 350 units. Scan/s: 4,51.

Identification of components was gained from retention times of SPB-1 and SupelcoWax-10 columns and their mass spectra. Retention times were compared with samples saved in laboratory database with more than 400 volatile natural compounds).

Relative amount of components was calculated according to GC areas without FID response factor correction.

4.4.2. Antifungal activity

4.4.2.1. Fungal strains

The antifungal activity of Lavandula angustifolia essential oil was tested on Candida, Dermatophyte and Aspergillus strains. The strains were obtained by several methods. Candida crusei H9 and Candida guillermondii MAT23 were isolated from recurrent cases of vulvovaginal candidosis, Candida albicans ATCC 10231, Candida tropicalis ATCC 13803 and Candida parapsilopsis ATCC 90018 were bought from American type Culture Collection. Cryptococcus neoformans CECT 1078 was gained from Colección Espanola de Cultivos Tipo. Filamentous fungi Aspergillus flavus F44 was isolated from bronchial secretion, Aspergillus niger ATCC 16404 and Aspergillus fumigatus 46645 were supplied by American Culture Type Collection. Dermatophytes Epidermophyton floccosum FF9, Trichophyton mentagrophytes FF7 and Microsporum canis FF1 were isolated from nails and skin and last dermatophytes Trichophyton rubrum CECT 2794, Trichophyton verrucosum CECT 2992, Trichophyton mentagrophytes var. interdigitale CECT 2958, Microsporum gypseum CECT 2908 were obtained by Collectión Espanola de Cultivos Tipo.

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Fungi were identified by standard microbiological methods. All strains were stored on Sabourad agar with 20 % of glycerol at -70 ˚C. Before the actual testing each isolate was inoculated on Sabourad agar to make sure that the strain is not contaminated and has standard grow characteristic.

4.4.2.2. Method

Antifungal activity was tested by macrodilution broth method (macrodilution was chosen for the possibility to test oil in glass tubes and so avoid reaction of oil with the plastic ones) to detect minimal inhibitory concentrations (MIC) and minimal lethal concentrations (MLC) of essential oil. The whole experiment was performed according to Clinical and Laboratory Standards Institute (CLSI) reference protocols M27-A3[75]

and M38-A2.[76]

Dilutions of the oil were prepared by serial dilution (the same amount of essential oil and DMSO were mixed, half of the mixture was added to the specific amount of medium, mixed and half of this new mixture was again transefered to the new medium and so on), concentration ranging from 0,08 to 20 µL/mL. Final concentration of DMSO did not exceed 2 % (v/v).

The inoculum suspensions were prepared from fungal strains diluting in PRMI 1640 broth in appropriate density of (1-2) x 10³ cells/ml for yeasts or (1-2) x 10⁴ cells/ml for filamentous fungi and placed in 12 x 75 mm glass test tubes. The cell density was then confirmed by counting on Sabourad agar.

Different essential oil concentrations were added to the test tubes, which were subsequently aerobically incubated at 35 ˚C for 48 hours for Candida spp. and Aspergillus spp, at 35 ˚C and 72 hours for Cryptococcus neoformans and at 30 ˚C for 7 days for Dermatophytes. The oil-free growth control and DMSO toxicity control tubes were used.

The minimal lethal concentration (MIC) was then evaluated to detect the lowest concentration of the oil which causes the full growth inhibition.

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To measure minimal lethal concentration (MLC), 20 µL aliquots were taken from each negative tube and the first positive tube (as a growth control) from the MIC reading and were cultured in Sabourad dextrose agar. Plates were incubated at 35 ˚C for 48 hours for Candida and Aspergillus, at 35 ˚C and 72 hours for Cryptococcus neoformans and for 30 ˚C for 7 days for dermatophytes. MLC values were determined as the lowest concentration of the oil causing fungal death.

Two reference antifungal compounds, amphotericin B (Fluka) and fluconazole (Pfizer, UK) were used as standard antifungal drugs for quality control. For all conditions the RPMI 1640 medium (containing L-glutamine, phenol red pH indicator and without bicarbonate) was used. The experiment was performed in triplicate.